The general goals of this research are the same as the previous proposal, to develop techniques for minimally invasive bubble generation, growth and maintenance in vivo. A specific application, diagnosis of urinary reflux has now been targeted for development and preliminary, complimentary investigations will be performed on a second medical applications, diagnosis of interstitial cystitis. The first grant period has been one in which almost all the necessary elements of noninvasive ureteral vesicular reflux diagnosis have been shown to be feasible. Some techniques of bolus generation for blood flow and perfusion imaging also have been demonstrated, but will not be pursued further here. For reflux, apparently noninvasive acoustic generation of bubbles has been demonstrated in the fresh, full dog bladder under conditions which should be more difficult than the in vivo case. In albumin-stabilized microbubbles, with still less than optimal techniques, we have demonstrated acoustically-induced growth in backscatter of as much as 15 dB in the 2-3 MHz frequency range wit a 0.5s, 1 MPa, swept-frequency pulse. More importantly, calculations of rectified diffusion of free bubbles show a 60 fold increase in backscatterat the peak frequency (in 1 sec, 0.4 MPa frequency priches), with great narrowing of the backscattered frequency distribution to 2.8% of the resonant frequency. Additionally our experiments with static depressurization show greatly restricted diffusion into albumin-stabilized microbubbles, indicating that we should be able to grow free bubbles in the bladder at much faster rates than we have now demonstrated in stabilized microbubbles. Acoustic trapping and movement of a limited quantity of bubbles to a simulated bladder orifice has been demonstrated as has trapping of bubbles in the beam in an in situ canine bladder. Finally, we have demonstrated in vivo that even early reflux can be diagnosed acoustically given adequate stabilized microbubbles in the bladder. This proposal is directed to careful, quantitative research to make ureteral vesicular reflux diagnosis easily workable. We propose to determine a simple and robust method of generating and characterizing copious quantities of bubbles in appropriately pure water, test the techniques on a wide range of urine properties in excised bladders representing in vivo conditions, and test the bubble generation and control and reflux diagnosis in an in vivo animal model. A search for acoustic/bubble conditions to cause bladder wall irritation in an animal model, with and without cystitis, will be undertaken to aid development of reflux diagnosis with low risk of immediate effects and to initiate investigation of possible methods of diagnosing interstitial cystitis.